2. A. Okra.
Okra (Abelmoschus esculentus) is one of the most important vegetable crops. The fruits are consumed in various forms in a number of countries. Okra has also been used as a source of fiber and for the production of oil and proteins.
Okra is susceptible to many insect pests and diseases which reduce the yield across the Okra growing regions. Okra yellow vein mosaic virus is a devastating disease in India and many other countries. This crop is extensively damaged by the Lepidopteran insect/pests viz.; Shoot and fruit borer (Earias vitella, E. insulana) and the Fruit borer (Helicoverpa armigera). Genetic improvement by conventional plant breeding is impaired due to the lack of resistance sources to pests and diseases in Okra germplasm.
2. B. Plant Cell and Tissue Culture
Each plant cell has the inherent ability for independent development into a whole organism if provided with the proper external conditions. Since the early demonstration of this ability, viz., totipotency and differentiation in vitro, plant tissue culture techniques have been widely used in the clonal multiplication of plants (Herberlandt., Sber. Akad. Wiss. Wien. (1902) 111:69-92).
Plant tissue culture technology is making significant contributions to agriculture in the clonal propagation, haploid breeding, mutant cultures, pathogen free plants, cryopreservation of plant tissues for the establishment of in vitro gene banks, production of secondary products and genetic engineering of plants (Chilton., Scientific American (1983) 248.6:36-45).
The prospects of success with the genetic engineering of plants have created considerable public interest. This technique involves the insertion of foreign genes into plant cells using vectors and the regeneration of whole plants from transformed single cells using plant tissue culture techniques. Although tissue culture based plant regeneration methods have been standardized for a wide variety of plant species, many crops have been recalcitrant and thus restricts the genetic engineering potential of these plants.
2. C. Tissue Culture of Okra
Tissue culture based direct plant regeneration of okra has been described by Mangat and Roy., Plant Science (1986) 47:57-62. This investigation outlines the comparative tissue culture responses of hypocotyl, cotyledon, cotyledonary node and primary leaf explants aseptically grown okra seedlings cultured on 6 different media. The plant regeneration was not attained from hypocotyl and leaf segment explants in all the 6 media tested. The cotyledon explants responded moderately in plant regeneration, on one out of 6 media tested. Cotyledon node explants did not regenerate on 3 of the media, whereas, responded low in shoot regeneration on 1 medium, and responded very high in the shoot regeneration on another medium.
In the above publication the shoot regeneration frequency is mentioned as nil, low, moderate and very high and not in figures (%). It makes the comparison of regeneration frequency, difficult.
In a second publication, Roy and Mangat (Roy et al., Plant Science (1989) 60:77-82) have reported the regeneration of plants from cotyledonary-axil derived callus tissue of Okra. Callus induction that resulted from all the explants cultured on MS medium supplemented with the benzyl adenine (BA). The hypocotyl-derived callus remained non-organogenic, whereas, cotyledonary-axil-derived callus produced shoots. The addition of silver nitrate in media resulted in up to 74% of the bud primordia going onto produce shoots.
The study of prior art shows no method available for the regeneration of plants from plumule of embryo. Our present inventions describe methods for the high efficiency plant regeneration from plumule of okra for the first time.
The study of prior art also shows no method available for the regeneration of plants from the other explants also tested during in our investigation.
2. D. Plant Transformation and Generation of Transgenic Plants
The development of gene transfer techniques for plant species is of great interest, importance and value because it can be used for the transfer of beneficial genes of interest into plants.
A variety of techniques have been used to introduce foreign genes into plant cells. Agrobacterium mediated transformation has been described by Murai et al., Science (1983) 222:476-482, Fraley et al., Proc. Natl. Acad. Sci. USA (1983) 80:4803-4807; Direct DNA uptake method has been described by Lorz et al., Mol. Gen. Genet., (1985) 199:178-182, Portrykus et al., Mol. Gen. Genet., (1985) 199:183-188; Microinjection method has been described by Crossway et al., Mol. Gen. Genet., (1986) 202:179-185; High velocity micro-projectile method has been described by Klein et al., Nature (1987) 327:70-73 and Electroporation method has been described by Fromm et al., Proc. Natl. Acad. Sci. USA (1985) 82:5824-5828, Fromm et al., Nature (1986) 319:791-793.
2. E. Agrobacterium-Mediated Transformation
One of the most common methods of introducing foreign genes into plant cells is through Agrobacterium-mediated transformation. Agrobacterium is a natural plant pathogen and it mediates genetic transformation as part of the natural process it utilizes when it infects a plant cell. During the process of transformation a specific segment of the vector which is known as T-DNA, is transferred into the cells. The T-DNA of Agrobacterium can be engineered to contain gene/s or DNA sequences of interest that can be transferred into the host plant cells and inserted into the plant genome.
Agrobacterium-mediated transformation is attractive because of the ease of the protocol coupled with minimal equipment costs. Moreover, transgenic plants obtained by this method often contain a single copy of T-DNA integrations.
Agrobacterium-mediated transformation and the subsequent regeneration of transgenic plants carrying inserted genes were described by Murai et al., Science (1983) 222:476-482. Fraley et al., Proc. Natl. Acad. Sci. USA (1983) 80:4803-4807. De Block et al., The EMBO Journal (1984) 3:1681-1689 and Horsch et al., Science (1985) 227:1229-1231.
2. F. Biolistic-Mediated or Particle Bombardment Method of Transformation
Another common method of introducing foreign gene/s into plant cells is using particle bombardment which is also known as biolistic or high velocity microprojectile. The basis of particle bombardment is the acceleration of particles coated with gene/s of interest toward cells, resulting in the penetration of the protoplasm by the particles and subsequent expression of the introduced gene/s. In this method helium pressure is used to accelerate particles coated with DNA into the cells.
Microprojectile bombardment can transform diverse target tissues. Particle bombardment and subsequent regeneration of transgenic plants carrying inserted genes were described by Klein et al., Nature (1987) 327:70-73. Klein et al., Bio/Technology (1992) 10:286-292. Casas et al., Proc. Natl. Acad. Sci. USA (1993) 90: 11212-11216.
2. G. Transformation of Okra
The study of prior art shows no method available for the transformation of Okra plant, cells and tissues. Our present inventions describe methods for the transient and stable transformation of okra plant, cells and tissues using Agrobacterium-mediated and biolistic transformation systems for the first time.
2. H. Marker Based Transformation Systems
In the marker based transformation system, the gene of interest and the selectable marker gene (Eg. NPT II gene) are linked. In the marker based Agrobacterium-mediated transformation system, the T-DNA is engineered to contain the gene of interest and the marker gene. Where as in the marker based biolistic transformation system the plasmid used, contains the gene of interest and the selectable marker gene. The transgenic plants generated from the above systems contain the marker gene along with the gene of interest.
2. I. Marker-Free Transformation Systems
Marker-free transformation systems have the advantages of introducing agronomical important genes, and at the same time, avoiding the introduction of the selectable marker genes. Different methods have been employed for the generation of marker-free transgenic plants. These methods include Agrobacterium-mediated co-transformation, excision of the selectable marker via crellox recombination, use of transposable elements, co-bombardment of the plasmids and altered metabolism (Yoder et al., Bio/Technology (1994) 12:263-267).
Agrobacterium-mediated co-transformation, using two separate plasmids in a single Agrobacterium, i.e. one vector carrying the selectable marker gene in one T-DNA and the other vector carrying the gene of interest in another T-DNA are used. The transgenic plants generated from this transformation system are analyzed and the plants having gene of interest but with out selectable marker gene are selected in the further generations from the segregated progenies (Komori et al., The Plant Journal (1996) 10: 165-174).
2. J. Engineering Plants for Insect Resistance
2. J. A. Bacillus thuringiensis (Bt) gene
Bacillus thuringiensis (Bt) is a gram positive bacterium which produces a variety of insecticidal crystal proteins toxic to insects. These Bt genes have been successfully engineered into crop plants to get resistance to the specific insect pests in a number of crops. For example insect resistant transgenic tomato plants were generated with Bt gene by Fischhoff et al., Bio/Technology (1987) 5:807-813.
2. J. B. Protease Inhibitors
Protease inhibitors are an important element of plant defense response to insect predation. Transgenic plants expressing protease inhibitors show enhanced resistance to predation by pests, indicating the useful function of these inhibitors (Johnson et al., PNAS (1990) 86:9871-9875.
2. K. Insect Bioassays
The bioassays using specific insect pests with plants/plant parts are conducted to understand the resistance or susceptibility of the plant towards the pest. The efficacy of the specific insect resistant protein expressed in transgenic plants is tested with the specific target pest in the insect bioassays. The resistance of the plant towards specific target pests is compared with non-transgenic plant controls.